Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in delta-aminolevulinic acid formation during chlorophyll biosynthesis.
The formation of delta-aminolevulinic acid, the first committed precursor in porphyrin biosynthesis, occurs in certain bacteria and in the chloroplasts of plants and algae in a three-step, tRNA-dependent transformation of glutamate. Glutamyl-tRNA reductase, the second enzyme of this pathway, reduces the activated carboxyl group of glutamyl-tRNA (Glu-tRNA) in the presence of NADPH and releases glutamate 1-semialdehyde (GSA). We have purified Glu-tRNA reductase from Chlamydomonas reinhardtii by employing six different chromatographic separations. The apparent molecular mass of the protein when analyzed under both denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 130,000 Da; this indicates that the active enzyme is a monomer. In the presence of NADPH Glu-tRNA reductase catalyzed the reduction to GSA of glutamate acylated to the homologous tRNA. Thus, the reductase alone is sufficient for conversion of Glu-tRNA to GSA. In the absence of NADPH, a stable complex of Glu-tRNA reductase with Glu-tRNA can be isolated.
Chen
M W
MW
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Jahn
D
D
O'Neill
G P
GP
Söll
D
D
D000622:Aminolevulinic Acid / Q000378:metabolism*; D002696:Chlamydomonas / Q000201:enzymology*; D002734:Chlorophyll / Q000096:biosynthesis*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005975:Glutamate-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D007982:Levulinic Acids / Q000378:metabolism*; D008970:Molecular Weight
D007982:Levulinic Acids; D002734:Chlorophyll; D000622:Aminolevulinic Acid; D007658:Ketone Oxidoreductases; C059846:glutamyl tRNA dehydrogenase; D005975:Glutamate-tRNA Ligase